Loss of Cdc42 in Exocrine Acini Decreases Saliva Secretion but Increases Tear Secretion-A Potential Model of Exocrine Gland Senescence.

Cdc42 is a small GTPase essential for the cell cycle, morphogenesis, and cell adhesion, and it is involved in the polarity of epithelial cells. However, the functional roles of Cdc42 in exocrine glands, such as the maintenance of acini and water secretion, are not yet well understood. In this study, we generated acinar-cell-specific Cdc42 conditional knockout (Cdc42cKO) mice to assess their maintenance of acinar cells and physiological functions in the salivary glands (SGs) and lacrimal glands (LGs). Our data revealed that the loss of Cdc42 altered the luminal structures to bulging structures and induced acinar cell apoptosis in both the parotid glands (PGs) and LGs of Cdc42cKO mice. Interestingly, saliva secretion in response to pilocarpine stimulation was decreased in the Cdc42cKO group, whereas tear secretion was increased. Consistent with the water secretion results, protein expression of the water channel AQP5 in acinar cells was also decreased in the PGs but conversely increased in the LGs. Moreover, the changes that increased AQP5 expression in LGs occurred in the acinar cells rather than the duct cells. The present study demonstrates that Cdc42 is involved in the structural and survival maintenance of acinar cells in SGs and LGs. On the other hand, depletion of Cdc42 caused the opposite physiological phenomena between PGs and LGs.

Outbreaks of Campylobacteriosis Caused by Drinking Raw Milk in Japan: Evidence of Relationship Between Milk and Patients by Using Whole Genome Sequencing.

Raw milk may contain some infectious bacteria and usually requires pasteurization before drinking. In this study, we report rare outbreaks of campylobacteriosis associated with raw milk in Japan, and the application of whole genome sequencing (WGS) to studies on foodborne diseases. In August 2018, there were three outbreaks of campylobacteriosis, presumably caused by the consumption of unpasteurized raw milk, derived from the same farm; thus, these three outbreaks seemed to be associated with a single contaminant at the farm. Therefore, we analyzed Campylobacter jejuni isolates obtained at the three locations using several genetic methods. The sequence type of each isolate, revealed by multilocus sequence typing, was ST-61, and the profile determined using pulsed-field gel electrophoresis was the same; however, neither method could distinguish these from previously obtained strains. Subsequently, we performed WGS and single nucleotide variant (SNV) analysis that provided evidence of clonality, indicating that C. jejuni contamination was attributed to the farm. As in this study, evidence suggests that SNV analysis provides molecular biological support in cases with sufficient epidemiological information. Hence, similar analytical methods may be used in other sporadic cases to elucidate the relevance of the cases.

A case of immunoglobulin G4-related retroperitoneal fibrosis and hypophysitis with antecedent respiratory disease followed by spontaneous remission and recurrence.

A 65-year-old man presented with apparent bronchopneumonia. After treatment with antibiotics, he showed eosinophilia. Computed tomography (CT) imaging revealed bilateral consolidation, ground-glass opacities with nodular consolidations, and pleural effusion. Lung biopsy showed organising pneumonia with lymphoplasmacytic infiltration in the alveolar septa and in the thickened pleura and interlobular septa. All pulmonary abnormalities spontaneously went into remission within 12 months. At 73 years old, a follow-up CT scan revealed small nodules in both lungs and the review of the head CT scan showed thickening of the pituitary stalk in studying prolonged headache. Two years later, he visited the hospital complaining of severe oedema on the lower extremities with high serum immunoglobulin (Ig)G4 186 mg/dl. A whole-body CT scan showed retroperitoneal mass surrounding aortic bifurcation and compressing inferior vena cava, pituitary stalk thickening and gland swelling, and enlarged pulmonary nodules. Anterior pituitary stimulation tests showed central hypothyroidism, central hypogonadism, and adult growth hormone deficiency with partial primary hypoadrenocorticism. Retroperitoneal mass biopsy showed storiform fibrosis and obliterative phlebitis with marked lymphoplasmacytic infiltration with moderate IgG4-positivity. Immunostaining of the former lung specimen revealed dense interstitial infiltration of IgG4-positive cells. These findings indicated metachronous development of IgG4-related disease in lung, hypophysis, and retroperitoneum, according to the recent comprehensive diagnostic criteria of IgG4-related disease. Glucocorticoid therapy ameliorated oedema, on the other hand, unmasked partial diabetes insipidus at the initial dose of the treatment. Hypothyroidism and retroperitoneal mass regressed at 6 months of the treatment. This case warns us that long-term follow-up from prodromal to remission is necessary for the treatment of IgG4-related disease.

Review of genes potentially related to hyposecretion in male non-obese diabetic (NOD) mice, a Sjögren's syndrome model.

BACKGROUND: Sjögren's syndrome (SS) is known to cause dry eyes and mouth due to inflammation of the lacrimal and salivary glands. However, some reports imply that other factors trigger dry eyes and mouth. We previously investigated various factors using RNA-sequencing analysis of lacrimal glands from male non-obese diabetic (NOD) mice, an SS model. In this review, we described (1) the exocrine features of male and female NOD mice, (2) the up- and down-regulated genes in the lacrimal glands of male NOD mice as revealed by our RNA-sequencing data, and (3) comparisons between these genes and data in the Salivary Gland Gene Expression Atlas. HIGHLIGHTS: Male NOD mice exhibit a steady worsening of lacrimal hyposecretion and dacryoadenitis, whereas females exhibit a complex pathophysiological condition that includes diabetic disease, salivary hyposecretion, and sialadenitis. Ctss, an up-regulated gene, is a potential inducer of lacrimal hyposecretion and is also expressed in salivary glands. Two other up-regulated genes, Ccl5 and Cxcl13, may worsen the inflammation of SS in both the lacrimal and salivary glands. The genes Esp23, Obp1a, and Spc25 were detected as down-regulated, but judging the relationship between these genes and hyposecretion is difficult as only limited information is available. Another down-regulated gene, Arg1, is involved in lacrimal hyposecretion, and it also has the potential to cause salivary hyposecretion in NOD mice. CONCLUSION: In NOD mice, males may be better than females at evaluating the pathophysiology of SS. Some regulated genes revealed by our RNA-sequencing data might be potential therapeutic targets for SS.

Blueberry Stem Extract Prevents Lacrimal Hyposecretion in Non-obese Diabetic Mice via Activation of AMPK.

BACKGROUND/AIM: Tears secreted from the lacrimal gland are essential for preserving the ocular surface. Thus, dysfunction of the lacrimal gland in Sjögren's syndrome (SS) can lead to dry eye, resulting in a reduced quality of life. We previously reported that blueberry 'leaf' water extract prevents lacrimal hyposecretion in male non-obese diabetic (NOD) mice in a SS-like model. In this study, we investigated the effect of blueberry 'stem' water extract (BStEx) on lacrimal hyposecretion in NOD mice. MATERIALS AND METHODS: Male NOD mice were fed 1% BStEx or control (AIN-93G) for 2, 4, or 6 weeks from 4 weeks of age. Pilocarpine-induced tear secretion was measured using a phenol red-impregnated thread. The lacrimal glands were histologically evaluated by HE staining. Inflammatory cytokine levels in the lacrimal glands were measured using ELISA. Immunostaining was performed to examine aquaporin 5 (AQP5) localization. The expression levels of autophagy-related proteins, AQP5, and phosphorylated AMPK were measured using western blotting. RESULTS: After feeding BStEx to mice for 4 or 6 weeks, tear volume was observed to have increased in the BStEx group compared with that in the control group. There were no significant differences in inflammatory cell infiltration, autophagy-related protein expression, or the localization and expression of AQP5 in the lacrimal glands between the two groups. In contrast, AMPK phosphorylation increased in the BStEx group. CONCLUSION: BStEx prevented lacrimal hyposecretion in the SS-like model of male NOD mice, probably by opening tight junctions via the activation of AMPK in lacrimal acinar cells.

Blueberry Leaf Extract Prevents Lacrimal Hyposecretion in Sjögren's Syndrome-like Model of Non-obese Diabetic Mice.

BACKGROUND/AIM: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. MATERIALS AND METHODS: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1ß and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. RESULTS: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. CONCLUSION: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.

Retinal damage alters gene expression profile in lacrimal glands of mice.

Early detection of such retinal diseases as glaucoma and age-related macular degeneration (AMD) is important to prevent blindness. There have been reports of changes in some components in the tears of glaucoma and AMD patients, suggesting tears' potential usefulness in screening for retinal diseases. We hypothesized that retinal damage might alter gene expression in the lacrimal gland, leading to those changes in tear components. We caused retinal damage in mice by intravitreal injection of N-methyl-d-aspartate (NMDA) or excessive light exposure. Hematoxylin and eosin staining showed no histological changes in the lacrimal glands of animals whose retinas had been damaged. However, RNA sequencing of lacrimal glands on the 3rd day after NMDA injection or light exposure revealed changes in the expression of 491 genes (268 up-regulated; 223 down-regulated) in the NMDA group and 531 genes (311 up-regulated; 220 down-regulated) in the light group. Further gene-set enrichment analysis indicated that both types of retinal damage activated the immune system in the lacrimal glands. This is the first demonstration that retinal damage can alter gene expression in the lacrimal glands, and it might lead to a novel non-invasive screening method for early detection of retinal diseases.

Cryopreservation of Biologically Functional Submandibular Gland Rudiments from Fetal Mice.

BACKGROUND/AIM: Cryopreservation of cell lines has been widely used in the laboratory; however, cryopreservation of organs is still considered to be difficult. The submandibular gland (SMG) of fetal mice is one of the best-characterized organs. We investigated the conditions for cryopreserving SMG rudiments. MATERIALS AND METHODS: Embryonic day 13 SMG rudiments were cryopreserved with or without a cryoprotectant. They were thawed and incubated in DMEM/F12 medium. Moreover, the influence of EGF stimulation on the signaling cascade after frozen-thawing the rudiments was analyzed by Western blotting. RESULTS: When SMG rudiments were cryopreserved without a cryoprotectant, all cells in the rudiments died. However, the SMG rudiments that had been preserved in a cryoprotectant showed branching morphogenesis. Additionally, the responsiveness of signaling cascades to EGF did not differ between frozen with a cryoprotectant and non-frozen rudiments. CONCLUSION: Cryopreservation might be a useful technology for preserving tissues from small organs, such as fetal SMG rudiments.

Arginase 1 is involved in lacrimal hyposecretion in male NOD mice, a model of Sjögren's syndrome, regardless of dacryoadenitis status.

KEY POINTS: Few reports have explored the possibility of involvement of non-inflammatory factors in lacrimal hyposecretion in Sjögren's syndrome (SS). RNA-sequencing analysis revealed that only four genes, including arginase 1, were downregulated in the lacrimal gland of SS model male mice (NOD mice) after onset of lacrimal hyposecretion and dacryoadenitis. Even in non-dacryoadenitis-type NOD mice, tear secretion and arginase 1 expression remained low. An arginase 1 inhibitor reduced tear secretion and partially reduced saliva secretion in BALB/c mice. The results indicate that a non-inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status. ABSTRACT: Lacrimal fluid (tears) is important for preservation of the ocular surface, and thus lacrimal hyposecretion in Sjögren's syndrome (SS) leads to reduced quality of life. However, the cause(s) of lacrimal hyposecretion remains unknown, even though many studies have been conducted from the perspective of inflammation. Here, we hypothesized that a non-inflammatory factor induces lacrimal hyposecretion in SS pathology, and to elucidate such a factor, we conducted transcriptome analysis of the lacrimal glands in male non-obese diabetic (NOD) mice as an SS model. The NOD mice showed inflammatory cell infiltration and decreased pilocarpine-induced tear secretion at and after 6 weeks of age compared to age-matched BALB/c mice. RNA-sequencing analysis revealed that only four genes, including arginase 1, were downregulated, whereas many genes relating to inflammation were upregulated, in the lacrimal glands of male NOD mice after onset of lacrimal hyposecretion and dacryoadenitis (lacrimal gland inflammation). Changes in the level of arginase 1 expression were confirmed by real-time RT-PCR and western blot analysis. Furthermore, non-dacryoadenitis-type NOD mice were used to investigate the relationships among arginase 1 expression, lacrimal hyposecretion and dacryoadenitis. Interestingly, these NOD mice retained the phenotype of dacryoadenitis with regard to tear secretion and arginase 1 expression level. An arginase 1 inhibitor reduced tear secretion and partially reduced saliva secretion in BALB/c mice. In conclusion, a non-inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status. These results shed light on the pathophysiological role of arginase 1 in SS (dry eye).

JPH203, a newly developed anti-cancer drug, shows a preincubation inhibitory effect on L-type amino acid transporter 1 function.

JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a primary role in the uptake of essential amino acids in tumor cells. Although a co-incubation inhibitory effect of JPH203 has been shown in a conventional uptake assay, its preincubation inhibitory effects have remained undetermined. Therefore, we aimed to characterize the preincubation inhibitory effects of JPH203 on LAT1 function using leucine uptake assays in LAT1-positive human colon cancer HT-29 cells. Preincubation of the cells with JPH203 (0.3 µM for 120 min) decreased the activity level to 30% of that in dimethylsulfoxide-treated cells. Similarly, in time-dependency analysis, preincubation of HT-29 cells with 10 µM JPH203 for 30, 60, and 120 min decreased the leucine uptake activity (42%, 32%, and 28% of that in control cells, respectively). Furthermore, the IC50 value of the combination of preincubation and co-incubation effects was lower than that of co-incubation inhibition alone (34.2 ± 3.6 nM vs. 99.2 ± 11.0 nM). In conclusion, we revealed that JPH203 has the capability to inhibit LAT1 function through preincubation effects. Moreover, preincubation synergistically enhances the co-incubation inhibitory effects. These findings provide a novel insight into the anti-cancer effects of JPH203 in cancer therapy.

Hypoosmotic stress induces flagellar biosynthesis and swimming motility in Escherichia albertii.

Bacteria use flagella as propellers to move to favorable environments. Escherichia albertii, a growing cause of foodborne illness and diarrhea, is reportedly non-motile and lacks flagella on its surface. Here, we report that 27 out of 59 E. albertii strains, collected mainly from humans and birds, showed swimming motility when cultured at low osmotic pressure. The biosynthesis of flagella in E. albertii cells was induced under ambient temperature and hypoosmotic pressure: conditions which resemble aquatic environments. Flagellar induction increased E. albertii survival in the intestinal epithelial cell culture containing gentamicin. Although genes involved in chemotaxis are not present in the E. albertii genome, the addition of glutamic acid, an amino acid known to regulate the internal cell osmolarity, augmented the proportion of swimming cells by 35-fold. These results suggest that flagellar biosynthesis and motility in E. albertii cells are controlled by their internal and external osmolarity.

Optimal dose of sunitinib for long-term treatment in Japanese patients with renal cell carcinoma.

BACKGROUND/AIM: Sunitinib is used for the treatment of metastatic renal cell carcinoma (mRCC). Asian patients, including Japanese, tend not to tolerate long-term sunitinib therapy of 50 mg p.o. once daily for 4 weeks, followed by 2 week off treatment due to severe adverse events at this dosage level. The aim of this retrospective study was to investigate the optimal dose of sunitinib for long-term continuation in Asian patients with mRCC. PATIENTS AND METHODS: The study cases were 50 patients with mRCC who were treated with sunitinib between June 2008 and December 2017. Risk analysis for "unacceptable" adverse events (depending on the physician, ranging from grade 2 to ≥ grade 3) leading to discontinuation of sunitinib was determined by time-dependent Cox proportional hazard regression analysis. RESULTS: A total of 54 unacceptable adverse events leading to discontinuation occurred. Multivariable analysis indicated that a sunitinib dose of ≤ 37.5 mg/day significantly reduced the risk of discontinuation due to adverse events in comparison with 50 mg/day [hazard ratio (HR) 0.08, 95% confidence interval (CI) 0.03-0.21, p < 0.001). The progression-free survival (PFS) with a sunitinib dose ≤ 37.5 mg/day was longer than that associated with a dose of 50 mg/day, albeit not to a statistically significant degree (120 days for ≤ 37.5 mg/day vs 41 days for 50 mg/day, HR 0.39, 95% CI 0.10-1.44, p = 0.157). CONCLUSION: Our findings suggest that the optimal dose of sunitinib for Asian, including Japanese, patients with mRCC is ≤ 37.5 mg/day.

Temozolomide has anti-tumor effects through the phosphorylation of cPLA2 on glioblastoma cells.

Temozolomide is an alkylating agent used as the first line of treatment for glioblastoma. However, chemoresistance to temozolomide is common in glioma patients. In addition, there are likely many unknown mechanisms for the anti-tumor effects of temozolomide. It is known that an alkylating agent, sulfur mustard, activates cytosolic phospholipase A2 (cPLA2) releasing arachidonic acid to suppress tumors. The present study was performed to elucidate the involvement of cPLA2 in the anti-tumor mechanisms of temozolomide. In three glioblastoma cell lines (GL261, U251MG and T98G), we performed several evaluations including cell viability, cell migration and apoptosis, to study temozolomide-induced anti-tumor effects. Further, we evaluated tumor size in the murine orthotropic glioblastoma model after oral administration of temozolomide. Finally, we investigated the phosphorylation of cPLA2 in GL261 cells treated with temozolomide, and clarified whether phosphorylation of cPLA2 affects cell growth. Temozolomide suppressed cell growth and cell migration in glioblastoma cells in vitro and showed anti-tumor effect in the murine orthotopic glioblastoma model in vivo. Furthermore, temozolomide increased phosphorylation of cPLA2, which was associated with suppression of cell growth. However, in MGMT high-expressing glioblastoma T98G cells, temozolomide could not suppress cell growth or cause phosphorylation of cPLA2. These findings indicate that temozolomide suppressed cell growth partly by phosphorylation of cPLA2 in glioblastoma cells. In addition, because temozolomide did not cause phosphorylation of cPLA2 in MGMT high-expressing glioblastoma T98G cells, phosphorylation of cPLA2 may be caused by DNA alkylation of temozolomide.

Sodium-coupled monocarboxylate transporter 1 interacts with the RING finger- and PDZ domain-containing protein PDZRN3.

Sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8) mediates monocarboxylate transport in the proximal tubule of the kidney. We have identified PDZK1 and PDZ domain-containing RING finger 3 (PDZRN3) as potent binding partners of SMCT1, which has a PDZ motif (Thr-Arg-Leu), by yeast two-hybrid screening and revealed that PDZK1 enhances the transport activity of SMCT1. In this study, we aimed to characterize the interaction between SMCT1 and PDZRN3 as well as to examine how PDZRN3 regulates SMCT1 function. An interaction between SMCT1 and PDZRN3 through the PDZ motif was observed in a co-immunoprecipitation assay and yeast two-hybrid assay. A transport assay showed that PDZRN3 abolished the enhancing effect of PDZK1 on nicotinate uptake via SMCT1. Our results suggest that SMCT1 interacts with PDZRN3 and that PDZRN3 may regulate SMCT1 function by interfering with the interaction between SMCT1 and PDZK1.

Rapid attainment of target trough concentrations of tacrolimus for early improvement of clinical symptoms in patients with ulcerative colitis.

WHAT IS KNOWN AND OBJECTIVE: The target trough concentration of tacrolimus for ulcerative colitis is recommended to be 10-15 ng/mL in the initial two weeks and 5-10 ng/mL in the later phase. However, the effectiveness of rapid attainment of these target trough concentrations of tacrolimus in patients with ulcerative colitis is still unclear. In the present study, we evaluated the clinical efficacy and safety of rapid attainment of target trough concentrations of tacrolimus in patients with ulcerative colitis. METHODS: A prospective cohort was conducted at Gifu University Hospital in Gifu, Japan. Hospitalized patients who received tacrolimus for the treatment of ulcerative colitis between April 2009 and March 2017 were enrolled. Since June 2011, the initial loading dose of tacrolimus increased from 0.05 to 0.1-0.2 mg/kg/d, and the maintenance dose to achieve the target trough concentration was determined to be 12.5 ng/mL by proportional calculation with measured blood concentration. The period required to attain target trough concentration and the clinical efficacy before and after dosage modification was compared. RESULTS: The initial dose after dosage modification was significantly increased compared to that before dosage modification (0.10 [0.04-0.22], median [range] mg/kg/d vs 0.05 [0.03-0.05] mg/kg/d, P < 0.001). The period required to attain a target trough concentration over 10 ng/mL was significantly shortened by dosage modification (6 [4-14] days before dosage modification vs 4.5 [2-8] days after modification, P = 0.048). Further, stool frequency score was significantly improved after dosage modification, without affecting the incidence of adverse events. WHAT IS NEW AND CONCLUSION: Our findings suggest that rapid attainment of the target trough concentration of tacrolimus improves clinical symptoms in patients with ulcerative colitis.

Time-Dependent Decline in Serum Phenytoin Concentration With Heightened Convulsive Seizure Risk by Prolonged Administration of Fosphenytoin in Japanese: A Retrospective Study.

BACKGROUND: Because clinical data to confirm the safety and effectiveness of fosphenytoin, a prodrug of phenytoin, are insufficient, the length of administration of fosphenytoin is restricted. Nevertheless, some cases require fosphenytoin administration for more than a few days. The aim of this study was to retrospectively investigate the serum concentration of phenytoin in adult Japanese patients who received intravenous fosphenytoin therapy for more than 3 days. METHODS: Patients injected with intravenous fosphenytoin for more than 3 days at Gifu University Hospital between January 2012 and September 2014 were enrolled. Individual pharmacokinetic parameters were predicted by Bayesian estimation using NONMEM software, and the maintenance dose of fosphenytoin required to maintain the therapeutic trough concentration (10-20 mcg/mL) was calculated from the parameters. RESULTS: Among a total of 8 patients, the serum trough concentration of phenytoin decreased with each day after repeated injection of fosphenytoin. The incidence rate of significant convulsive seizures was increased time dependently (0% on day 1, 12.5% on day 2, 25% on day 3, and 66.7% on day 4 and after). Phenytoin clearance showed a time-dependent increase. The maintenance dose of fosphenytoin required to maintain the therapeutic trough concentration was simulated to be 779.8 ± 316.8 mg/d, a dose that was markedly higher than the actual maintenance dose (414.1 ± 55.7 mg/d). CONCLUSIONS: Prolonged use of fosphenytoin, for such patients as those with autoimmune-mediated encephalopathy accompanied with reflux disease and/or ileus, time dependently decreased the serum concentration of phenytoin and increased the risk of convulsion. Therefore, the maintenance dose should be increased to maintain the therapeutic serum concentration.

Evaluation of the Microbiological Efficacy of a Single 2-Gram Dose of Extended-Release Azithromycin by Population Pharmacokinetics and Simulation in Japanese Patients with Gonococcal Urethritis.

The objective of this study was to analyze the relationship between the pharmacokinetic (PK)/pharmacodynamic (PD) parameters of a single 2-g dose of extended-release formulation of azithromycin (AZM-SR) and its microbiological efficacy against gonococcal urethritis. Fifty male patients with gonococcal urethritis were enrolled in this study. In 36 patients, the plasma AZM concentrations were measured using liquid chromatography-tandem mass spectrometry, the AZM MIC values for the Neisseria gonorrhoeae isolates were determined, and the microbiological outcomes were assessed. AZM-SR monotherapy eradicated N. gonorrhoeae in 30 (83%) of the 36 patients. AZM MICs ranged from 0.03 to 2 mg/liter. The mean value of the area under the concentration-time curve (AUC), estimated by population PK analysis using a two-compartment model, was 20.8 mg · h/liter. Logistic regression analysis showed that the PK/PD target value required to predict an N. gonorrhoeae eradication rate of ≥95% was a calculated AUC/MIC of ≥59.5. The AUC/MIC value was significantly higher in patients who achieved microbiological cure than in patients who achieved microbiological failure. Monte Carlo simulation using this MIC distribution revealed that the probability that AZM-SR monotherapy would produce an AUC/MIC exceeding the AUC/MIC target of 59.5 was 47%. Furthermore, the MIC distribution for strains isolated in this study was mostly consistent with that for strains currently circulating in Japan. In conclusion, in Japan, AZM-SR monotherapy may not be effective against gonococcal urethritis. Therefore, use of a single 2-g dose of AZM-SR either with or without other antibiotics could be an option to treat gonococcal urethritis if patients are allergic to ceftriaxone and spectinomycin or are diagnosed to be infected with an AZM-sensitive strain.

Involvement of cannabinoid receptor type 2 in light-induced degeneration of cells from mouse retinal cell line in vitro and mouse photoreceptors in vivo.

Earlier studies showed that the expressions of the agonists of the cannabinoid receptors are reduced in the vitreous humor of patients with age-related macular degeneration (AMD), and the cannabinoid type 2 receptor is present in the retinas of rats and monkeys. The purpose of this study was to determine whether the cannabinoid type 2 receptor is involved in the light-induced death of cultured 661W cells, an immortalized murine retinal cell line, and in the light-induced retinal degeneration in mice. Time-dependent changes in the expression and location of retinal cannabinoid type 2 receptor were determined by Western blot and immunostaining. The cannabinoid type 2 receptor was down-regulated in murine retinae and cone cells. In the in vitro studies, HU-308, a cannabinoid type 2 receptor agonist, had a protective effect on the light-induced death of 661W cells, and this effect was attenuated by SR144528, a cannabinoid type 2 receptor antagonist. Because the cannabinoid type 2 receptor is a G-protein coupled receptor and is coupled with Gi/o protein, we investigated the effects of the cAMP-dependent protein kinase (PKA). HU-308 and H89, a PKA inhibitor, deactivated PKA in retinal cone cells, and H89 also suppressed light-induced cell death. For the in vivo studies, a cannabinoid type 2 receptor agonist, HU-308, or an antagonist, SR144528, was injected intravitreally into mouse eyes before the light exposure. Electroretinography was used to determine the physiological status of the retinas. Injection of HU-308 improved the a- and b-waves of the ERGs and also the thickness of the outer nuclear layer of the murine retina after light exposure. These findings indicate that the cannabinoid type 2 receptor is involved in the light-induced retinal damage through PKA signaling. Thus, activation of cannabinoid type 2 receptor may be a therapeutic approach for light-associated retinal diseases.

Characterization of an L-Carnitine Transport System in Murine Photoreceptor Cell Line.

While it is well known that L-carnitine [3-hydroxy-4-(trimethylazaniumyl)-butanoate] is an essential molecule for ß-oxidation, it provides anti-oxidative effects as well. Since these effects have been observed in photoreceptor cells, the carnitine's intracellular concentration is considered to play a protective role against oxidative damage to those cells. However, even though its high hydrophilicity makes it likely that carnitine import is accomplished via a dedicated host transport system, the specific uptake process into those cells is currently unknown. Therefore, in this study, we sought to identify and characterize photoreceptor cell carnitine uptake transporter(s) utilizing 661W cells as a photoreceptor cell model. The results of our uptake assays showed that carnitine was transported into 661W cells in a saturable manner (Km=5.5 mM), and that the activity was susceptible to extracellular pH and Na+. While these data suggest the involvement of a transporter in 661W cell carnitine uptake, the observed transport profile did not correspond to any of the currently known carnitine transporters such as organic cation/carnitine transporter 1 (Octn1), Octn2, Octn3, B0,+ and Ct2. In fact, in our experiments, the mRNA expressions for such carnitine transporters in 661W cells were consistently very low and the carnitine transporter substrates did not inhibit the uptake activities. Taken as a whole, our results indicate that carnitine is transported into 661W cells in a carrier-mediated manner. However, since its transport modes cannot be fully explained by known carnitine transporters, it is highly likely that photoreceptor cells utilize a unique molecularly-based carnitine uptake system.

Rimonabant, a selective cannabinoid1 receptor antagonist, protects against light-induced retinal degeneration in vitro and in vivo.

The endocannabinoid system is involved in some neurodegenerative diseases such as Alzheimer's disease. An endogenous constellation of proteins related to cannabinoid1 receptor signaling, including free fatty acids, diacylglycerol lipase, and N-acylethanolamine-hydrolyzing acid amidase, are localized in the murine retina. Moreover, the expression levels of endogenous agonists of cannabinoid receptors are changed in the vitreous fluid. However, the role of the endocannabinoid system in the retina, particularly in the light-induced photoreceptor degeneration, remains unknown. Therefore, we investigated involvement of the cannabinoid1 receptor in light-induced retinal degeneration using in vitro and in vivo models. To evaluate the effect of cannabinoid1 receptors in light irradiation-induced cell death, the mouse retinal cone-cell line (661W) was treated with a cannabinoid1 receptor antagonist, rimonabant. Time-dependent changes of expression and localization of retinal cannabinoid1 receptors were measured using Western blot and immunostaining. Retinal damage was induced in mice by exposure to light, followed by intravitreal injection of rimonabant. Electroretinograms and histologic analyses were performed. Rimonabant suppressed light-induced photoreceptor cell death. Cannabinoid1 receptor expression was upregulated by light exposure. Treatment with rimonabant improved both a- and b-wave amplitudes and the thickness of the outer nuclear layer. These results suggest that the cannabinoid1 receptor is involved in light-induced retinal degeneration and it may represent a therapeutic target in the light-induced photoreceptor degeneration related diseases.